A laboratory study was conducted to estimate utilization rates of thiamine (vitamin B1) and lipid in whole fish, muscle, and liver tissues of fasting Chinook salmon (Oncorhynchus tshawytscha). The experiment was conducted with Chinook salmon held at 5ºC over a period of 150 days to simulate fasting during migration or overwintering. Chinook salmon body length and wet weight did not change (p > 0.05) over the course of the study; however, significant declines were observed in muscle thiamine (p < 0.01) and lipid (p < 0.01). There was an inverse relationship between lipid and water content. Under the experimental conditions with no strenuous swimming, thiamine utilization rates reported are conservative estimates and were found to be 5.3–6.8 pmol·g-1·day-1 in muscle tissue and ~110 pmol·g-1·day-1 in liver tissue over the first 100 days. Fasting lipid depletion rates in whole fish were calculated to be 0.14–0.16%·d-1. Muscle lipid decline rate (0.13%·day-1) over the first 100 days was similar to whole fish lipid loss, however, muscle lipid utilization was slower (0.04%·day-1) over the last 50 days. During periods of fasting, Chinook salmon deplete bodily reserves of both thiamine and lipid which may have consequences for successful spawning migration and overwinter survival.